The objectives of this proposal are to determine how protein kinase C epsilon (PKC5) sensitizes skin to ultraviolet radiation (UVR)-induced constitutive activation of Stat3 as well as the development of squamous cell carcinoma (SCC). Constitutive activation of Stat3 is an important component of the mechanism of skin tumor promotion and is associated with the development of human cancers including SCC. SCC, the non-melanoma form of human skin cancer, is metastatic. Chronic exposure to UVR is the major etiologic factor linked to the development of SCC. We have previously reported that PKC5 levels in mouse epidermis dictates the susceptibility of mice to the development of SCC by UVR. We now found that, as compared with wild-type mice, PKC5 transgenic mice, when exposed to UVR elicited constitutive phosphorylation of Stat3 at both tyrosine 705 and serine 727 residues. Also, PKC5 interacted with Stat3 and phosphorylated Stat3 at serine 727. PKC5 interaction with Stat3 was dependent on UVR treatment and was PKC5 isoform specific. Stat3 is activated by phosphorylation of a conserved tyrosine 705, which enables dimerization, nuclear translocation, and DNA binding. However, for maximum transcriptional activity, Stat3 also requires phosphorylation of Ser727. However, it is unclear to what extent such interactions of PKC5 with Stat3 are important for optimal Stat3 transactivation and UVR carcinogenicity in intact mouse skin in vivo and these investigations form the focus of this proposal. We hypothesize that in intact skin in vivo: 1) UVR-induced phosphorylation of Stat3Ser727 is an important component of the mechanism by which PKC5 imparts sensitivity to UVR induced development of SCC. 2) PKC5 directly interacts with Stat3 to phosphorylate Stat3Ser727 and regulates its transcriptional activity. 3) Several proteins including anchoring and scaffold proteins interact with PKC5 and Stat3 to ensure specificity in their signal transduction pathways. We propose three specific aims to test these hypotheses. 1: To determine, by knockin mutation, that Stat3Ser727 phosphorylation is essential for UVR- induced transcriptional activation of Stat3 and the development of SCC in PKC5 overexpressing transgenic mice. 2: To determine whether PKC5 directly interacts with Stat3. The complementary experiments, which may provide clues of PKC5-Stat3 direct interaction, will include: i) GST pull-down assay involving purified recombinant GST-fusion PKC5 and Stat3. ii) Assessment of the binding of purified recombinant PKC5 to Stat3 iii) Mapping interacting domains of PKC5 and Stat3. 3: To identify PKC5 and Stat3 interacting proteins. The goal of this specific aim is to determine whether PKC5 interaction with Stat3 involves an assembly of a multi- protein complex. PKC5 and Stat3 interacting proteins will be determined by immunoprecipitation followed by 2-D gel electrophoresis and proteomic analyses. Knowledge obtained will determine whether PKC5 and PKC5- interacting proteins including Stat3 are molecular targets for prevention and/or treatment of human SCC.7.